Our eyesight normally allows us to see things in three dimensions: width,
height and depth. This is known as stereoscopic vision or commonly as 3D. The
ability to perceive depth, volume and the distance between objects is important.
When we look at a photo, television or a film we can only see things in only two
dimensions. In the same way, high-powered microscopes lack any depth of field,
so objects appear flat. In this article I describe how to convert these
instruments to produce stereoscopic images.

In order to perceive depth it is necessary for one eye to make the
observation at an angle to the other. When this happens the brain fuses the two
different images into a single stereoscopic one. It is the slight difference in
angle that creates the impression of volume and depth. Normal microscopes can
magnify between 50 and 1200X using a single objective at a time. The majority of
these instruments are used in Biology, whilst others are used in metallurgy,
mineralogy and other areas. The simplest models are equipped with a single
eyepiece, whilst more complex one have binocular eyepieces and may be equipped
to use a camera. Even though they have two eyepieces each provides the eye with
identical images because a prism in fact splits a single image, thus the image
appears completely flat.

Many people would like to have the advantages that three-dimensional images
produced by stereoscopic microscopes offer. In the observation of protists it
enables one to follow their movements as they change depth and to appreciate the
internal disposition of organelles. This article describes how it is possible to
produce these effects with a normal microscope using a single objective.

Before describing the methods of high-power stereoscopic observation it is
necessary to say something about Köhler Illumination which is the more diffuse
illumination system in the microscopes. Figure 2 shows that the lamp filament and
the specimen come into focus at different levels along the ray path, so when the
specimen is in focus the filament is not and vice versa. This avoids
superimposing the two images and the specimen is evenly illuminated. In the
diagram the two paths are separated for clarity. Note that in the illustration the image of the specimen is focused near to the eyepiece,
however during the observations the light that comes out of the eyepiece is
parallel and the image is focused at
infinity.

The image of filters used must never come into focus at the same level as
that of the specimen, but must always be displaced. From the theoretical
viewpoint the best position for these filters is at the aperture diaphragm.
However, this position is in practice inconvenient, so they have to be
positioned between aperture diaphragm and the field diaphragm. Be aware that if
the filters are placed too close the field diaphragm their image will come into
focus on the specimen. The most convenient position tends to be just above the
illuminator, though this can be rather too close to the field diaphragm. During
this article this is the position that is referred to because it is convenient
and usually works well. Adjusting the position of the condenser can create a
situation where the filters are out of focus and the specimen is clearly
visible. However, if there is insufficient separation between the filter images
and the specimen mount the filters on a tubular support to distance them from
the field diaphragm.

The first technique that produced three-dimensional images
required the use of two polarizing filters arranged on the illuminator so that
horizontally polarized light attains an half of the objective and vertically
polarized light attains the other half of the objective. Then polarizing filters
were placed in the eyepieces of a binocular head so that each eyepiece only
allowed the rays polarized in one direction to pass. Each eyes thus received two
different images seen from a slightly different angle making stereoscopic vision
possible. Due to absorption by the polarizing
filters, it is also necessary to have an illumination system of a certain power.

Sheets of polarizing
plastic can readily be obtained from photographic shops or in this website:
http://www.3dlens.com/ (Product No:
#P210, Polarizing efficiency: 99.98%). Cut two strips that are polarized at
90° to each other and mount them above the illuminator as shown in Figures 3
and 5 without superimposing them.
The line of contact between the two filters must be orientated vertically
(figures 3 and 6). The two filters can be mounted using screws or glued with
silicone. Cut away the excess parts.

Place a polarizing filter on each eyepiece (figure 4). For the initial
trials these can be cut from the same sheet as the other two and can be held
on the eyepiece by adhesive tape. Later optical quality filters can replace
those on the eyepieces, but the others suffice because of their position
close to the lamp. Ones made from polarizing plastic are more than adequate
here.

You may be able to obtain the correct-sized filters to be placed on the
eyepieces from well-stocked photographic supplier or from the USA - Edmund Optics -http://www.edmundoptics.com/ .
Once you obtained these filters, mount them on the eyepieces.
The filters placed on the eyepieces must be orientated in such a way as to allow
light polarized in one sense to pass through one of them and light polarized in
the opposite sense to pass through the other. We will now see how to do this.

Now, having fixed the filters to the illuminator and eyepieces, switch on
the illuminator and put a slide containing freshly-collected protists on
the stage. Using the 20X objective lens adjust the height of the condenser so the
contact line between the two filters is in focus and central in both
eyepieces' field of view. No unpolarized light must pass between the two
lower filters, they can be overlapped by a fraction of a millimeter.

Rotate the polarizing filter
of the right eyepiece until the left-hand side of the field is obscured as
much as possible. Do the same thing with the filter on the left eyepiece,
this time obscuring the right-hand side of the field.

Now adjust the condenser again to obtain optimum illumination and to move the contact line out
of focus. You will see that the field becomes bright again. At this point, you should be able to see in
three exciting dimensions. If the specimen appears in an opposite relief (pseudoscopy), just rotate the filters
on the illuminator through 180°
to obtain the correct image.

Figure 5 - Polarizing filters
and their mount.

Figure 6 - Polarizing filters
placed on the illuminator.

Adjust the illumination level and the diaphragm, after which you can begin
your observations. As required change magnification, then adjust the diaphragm,
illumination and the height of the condenser. Increasing the magnification also
increases the stereoscopic effect and between 200 and 400X provides the best
conditions.

This method uses one half of the objective to observe the specimen from one
direction and the other half for the other direction. In order to achieve this,
the image of the polarizing filters must be in focus at the plane of rear focal
point of the objective (figure 2). This will occur precisely when the filters
are arranged at the aperture diaphragm. In reality, such precision is not
required. What is important is that when the specimen is in focus, the filters
are well out of focus. If the dust on the illuminator
comes into focus along with the sample, use a tube to raise the half-moon
filters a couple of centimetres. Once the initial trials
are complete the filters may be cut into semicircles to fit a ring constructed
to hold them snugly on top of the illuminator.

I have obtained excellent results with this method and the smear of water
under a coverslip, normally only a few tenths of a millimeter thick appears to
be a pool several meters deep. Unfortunately, for different reasons observation
in 3D using the polarizing filter technique doesn’t always work. If you fail to
obtain noticeable results check that there are no other polarizing filters left
on the condenser or elsewhere along the optical path. Sometimes one optical
component of the binocular box may interfere with the polarization of the light.
Some prisms and non-metallic mirrors have this effect. Observation in 3D of
permanent preparations often doesn’t yield good results.

If you are about to buy a
microscope and you want to know if you can adapt it for stereoscopy, do as
follows: take with you two cuttings of polarizing plastic. Switch on the
microscope and place one of the pieces of plastic at the exit of the
illuminator, so as to cover it completely. Place the other piece on top of the
right eyepiece and by rotating it verify that there is a point in which there is
a discrete extinction of the light. Do the same thing with the other eyepiece.

I have only used this technique for observing protistans and have to say
that I'm not tired of observing them yet. To finally see the form of an
Amoeba as it extends it pseudopodia upwards is inspiring. What can I say about
Ciliates that rise and descend through a forest of cyanobacteria? It is a real
joy to see Euplotes as it explores a strand of Spirogyra, passing under then
over it like a gigantic reed. Now I actually see it rise and sink, instead of
just moving in and out of focus! The interior of a Vorticella, with the
movements of its food vacuoles and the contractile vacuoles, is wonderful. A
friend, who is passionate about Diatoms, told us that our method allowed him
finally to appreciate their three-dimensional structure and it had
revolutionized his observation methods. This technique also increases the depth
of field because the eyes can accommodate more comfortably having the impression
of depth.

It is impossible to list every case where there might be an aesthetic or
practical advantage in having the third dimension at high magnification. In
order to see the advantages that this technique offers, try comparing it with
standard observations in different cases. Stereoscopy particularly offers
advantages when there is a lot of detritus present because they do not appear as
a single clump as they normally do, but actually lie at different levels and out
of focus. It is so spectacular to observe protists in three dimensions that once
you have tried this technique you will not be able to do without it.
This technique is useful for direct observation, but unfortunately it does not
lend itself to photography or filming in 3D.

This method involves first closing one half of the objective and then the
other whilst taking the two photos. It is useful for photographing stationary
objects. Actually it is not essential to close the objective, only to intercept
half of the beam by placing a piece of card on the diaphragm or more
conveniently, on the illuminator. Figure 2 shows how this corresponds with the
partial occlusion of the objective. The stereograms have to be observed through
a stereoscope or a 3D slide viewer. Alternatively they can be used to obtain an
anaglyph to be observed with red-cyan spectacles. Obviously this technique
does not lend itself to direct observation.

Like the previous technique, this method allows photography of stationary
objects and does not permit direct observation. It uses horizontal displacement
of the slide between two photographs, so that one becomes the left image and the
other is right partner. It has the advantage of maintaining optimum definition
and the colors of the image.

To observe the adjacent picture of Spirogyra try to superimpose the two photos
whilst keeping the eyes parallel. In order to do this, gradually move closer to
the screen until the two pictures fuse into a single, out-of-focus stereogram.
Move away slowly, trying to keep the images superimposed until the image becomes
clear. If you can cross your eyes easily, look at the second and third
stereograms whilst trying to look at the left picture with the right eye and
vice versa.

Some condensers can be de-centralized, that is they can be displaced from the
principal optical axis. This results in angled rays illuminating the specimen.
In order to obtain a stereoscopic image it is necessary to take two photos, one
with the condenser displaced leftwards and the second with is moved to the right.
In a similar way, the mirror can be slightly rightwards and then to the left on
microscopes with a moveable mirror, so the specimen is illuminated from
different angles for each photo.

The following procedure allows the direct observation, photography or
filming of moving objects to be done relatively easily. I describe a method
whilst not devoid of problems offers certain clear advantages. The technique
is similar to that based on polarizing filters except that it uses colored
filters instead. A red and a cyan filter are placed adjacently on the
illuminator (figure 11), cyan lies halfway between green and blue. The images
obtained by this method are called "anaglyphs" (superimposed stereograms)
and the images show characteristic red-cyan colors. The photos must be
observed through red-cyan spectacles. By convention the red filter is on
the left which results in the left eye receiving a slightly different image
from the right.

Obtain two pairs of bi-colored spectacles from photographic suppliers.
Remove the filters from one pair and place them on the illuminator. Put on
the second pair and look through the binocular microscope. When this is done
correctly it is possible to see the specimen in three dimensions. If the
specimen appears in opposite relief, change over the filters on the illuminator.

Some people are rather prejudiced against this technique
because of the image's modified colors and the inconvenience of wearing the
red-cyan spectacles. I have to say that it isn't really that bad because
one becomes used to wearing the glasses and the colors don't appear so
garish after a short time, rather they become greyish. Surprisingly, the
colors often appear quite normal, not quite as true as with normal
observation or using polarizing filters, nevertheless it is nowhere as bad
as you might imagine.

As I have said, this method has the great advantage
of allowing observation of moving specimens to be made easily. They can be
filmed in 3D and easily projected, the impression of three dimensions being
contained in a single image. This means that a single shot with one camera
is sufficient, whereas when polarizing filters are used two synchronized
cameras are needed. If you are limited to direct observation then the
polarizing filters method is certainly the best.

The colored filters method requires a binocular microscope, but to take
photos a simple student's monocular microscope is adequate. For microscopes
equipped with a mirror, it is necessary to obtain colored plastic films, as
near to the color of the spectacles as possible, and place them in front of
an illuminator with an opalescent bulb. Figure 12 shows how to do this with
a sheet of card.

Unlike the polarizing method, a video camera can be mounted on the
microscope and moving 3D images can be examined on a monitor or TV. This
enables the image to be observed by an entire class as long as red-cyan
spectacles are available.

A weak current can convert a transparent liquid crystal screen
from transparent to black. Using this principle it is possible to make 3D films
with just one video camera. A pair of liquid crystal screens placed on the
illuminator, in the same way as the polarizing or colored filters, can
alternately allow light to pass from one side of the illuminator or the other.
As we have seen, this corresponds to using one half of the objective and then
the other. By synchronizing the liquid crystal screens with the frames of the
video camera, alternate frames correspond with left and right halves of the
objective. In order to see these images in 3D it is necessary to use special
liquid crystal spectacles synchronized with the projection system.
Several research laboratories are beginning to offer TV screens that allow 3D
recordings to be shown without the need to wear spectacles.

As is well known, any increase in magnification causes a corresponding
decrease in depth of field. This limitation is particularly obvious when making
photographs, but is less so in direct observation when it is possible to change
focus to look at different levels. As a remedy for this poor depth of field it
is possible to superimpose several photographs, but the images produced in this
way are not sharp. Recently, programs have been produced that recover the
clear part of each photograph.

These techniques of making stereoscopic observations and photographs at high
magnifications will open up new horizons in your use of microscopes. I am sure
that once you have tried them you will find the methods hard to give up,
especially for the observation of protists. These techniques are within the
scope of any amateur microscopist. However, these
methods need a lot of trials and adjustments to produce satisfactory results.
The optics of microscopes, stereoscopy, polarization and anaglyphs are vast
fields and cannot really be fully described by the schematic information that I
have supplied. An understanding of optics will significantly improve your
ability to obtain good results from our techniques. Much supporting information
is readily available on the Internet.

The sight of microorganisms in pond water swimming up and down seen in 3D at
high magnifications is incredibly fascinating. You will not see anymore
Spirogyra as a mass of matted strands, but as separate fibers lying at
different levels sloping up or downwards with their elegant helical chloroplasts
inside each cell. At last, you will see if that strands have left-handed or
right-handed spirals. You will notice that the quality of observation is
noticeably improved because these techniques not only offer an aesthetic
improvement, but the addition of the third dimension provides further
information about the shape, size and position of the objects and organisms that
you are observing. All this helps you to understand them better.